摘要
目的应用基因克隆技术构建雌激素受体β1(ERβ1)及其5-8外显子缺失变异体ERβ△5-8(文中简称ERβ△),为ERβ功能的研究奠定基础。方法组织中提取总RNA,RT-PCR法扩增基因ERβ1及ERβ△,限制性酶切并克隆于以带EGFP绿色荧光基因及Myc报告基因的非融合真核细胞表达质粒IRES2-EGFP,菌群转化扩增后琼脂糖凝胶电泳,限制性酶切后行DNA测序鉴定。结果 PCR扩增及限制性酶切法证实重组真核表达质粒的构建成功。测序表明ERβ1长度为1593 bp,ERβ△长度为972 bp,与GeneBank上公布的蛋白编码区序列一致。结论成功构建ERβ1基因及其剪切变异体ERβ△真核表达载体。
Objective To construct eukaryotic expression plasmid of human estrogen receptorβ1（ERβ1） and the 5th to 8th exon missing variant ERβ△ 5-8（in this paper referred to as ERβ△ ）. Methods Genomic fragment of ER β1 and ERβ△ was amplified by RT-PCR. The recombinant plasmid was cleared by restriction endonuclease and cloned into eukaryotic expression vector IRES2-EGFP, which carried EGFP gene and Myc report gene and then transformed into the E.eoli DH5 a. The recombinant plasmid was identified by agarose gel electrophoresis. The genomie fragment of ER β1 and ERβ△ was determined and analyzed by DNA sequencing. Results Eukaryotic expression plasmid was identified by PCR and restriction endonuclease. Sequencing results show that the length of fragments is the same with the CSD of ER β 1 and its variant in the GeneBank. Conclusion This study successfully constructs the eukaryotic cell express cloning IRES2-EGFP-ER β 1 and IRES2-EGFP-ERβ△ .
出处
《中华普通外科学文献（电子版）》
2013年第4期18-20,共3页
Chinese Archives of General Surgery(Electronic Edition)
基金
广东省科技计划项目(2010B031600294)
